Vertical gel slab electrophoresis apparatus and method

ABSTRACT

A gel electrophoresis device in which the separating gel is cast in a vertical position between hydrophilic plates which form a gel slab chamber filled with a separating gel solution from the bottom for reducing fill turbulence allowing formation of a gel slab having a predetermined gradient. A removable seal is applied to the bottom of the gel slab chamber for containing the separating gel solution prior to polymerization. One gel slab chamber may be removably attached to each vertical side of a central temperature control chamber which is completely enclosed and which has an inlet and an outlet channel for circulation of temperature control fluid therethrough. An upper buffer chamber which opens upwardly is formed above the central temperature control chamber in communication with the top of the vertical gel slab chambers. The top of the gel slab is formed using a well forming implement made of polymerization inhibiting material so that the implement may be withdrawn after polymerization without altering the desired shape of the top of the vertical gel slab. The gel slab chambers attached to the central enclosed temperature control and upper buffer chamber are lowered into an outer chamber after polymerization of the separating gel solution and removal of the seals from the lower end of the gel slab chambers. The outer container is filled with a buffer solution in consequent directi contact with the lower end of the gel slabs, and the upper buffer chamber is filled with a buffer solution in direct contact with the top of the gel slabs. Samples are underlaid beneath the upper buffer solution atop the shaped upper surface of the gel slabs and a cover is placed on the outer container to contact a pair of electrodes which are in separate electrical contact with one of the upper and lower buffer chambers for applying a voltage therebetween, thereby causing electrophoretic separation of the sample within the gel slab.

This is a division of application Ser. No. 455,871 filed Mar. 28, 1975now U.S. Pat. No. 3,932,265.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a vertical gel slab formation andelectrophoresis apparatus, and more particularly to an apparatus inwhich gel slabs are formed in a gel slab chamber oriented in a truevertical position, having hydrophilic side walls, and which is filledfrom the bottom for eliminating turbulence and preserving separating gelsolution gradients.

2. Description of the prior art

Most prior slab electrophoresis devices do not provide for verticalcasting of the gel slab thereby trapping bubbles within the gel slabwhich distorts the separation of the sample therein duringelectrophoresis. Those devices providing for vertical casting of the gelslab do not have means for orienting the gel slab chamber in a truevertical position prior to polymerization and are all filled with theseparating gel solution from the top of the gel slab chamber therebyincreasing the probability of bubble formation and turbulence duringfilling which destroys or distorts any desired separating gel gradient.Gel slabs often allow leakage of buffer solution between the gel slaband the sides of the gel slab chamber, thereby short circuiting the gelslab and distorting the desired separation of the sample within the slabduring electrophoresis. Moreover, low concentration separating gelsolutions were often distorted due to a disparity of vertical forces atthe bottom of the gel slab arising from different fluid pressure headsin the gel slab chamber and the lower buffer chamber. Operator exposureto high voltages utilized during electrophoresis is possible in priorart devices.

A vertical gel slab formation and electrophoresis apparatus is thereforeneeded which will allow casting of gel slabs having predetermined gelgradients, eliminate deformation within low concentration gel slabs,prevent leakage of buffer solution through the gel slab chamber, andprovide operator protection from electrical shock.

SUMMARY OF THE INVENTION AND OBJECTS

An apparatus is disclosed which allows vertical formation of a gel slabfor use in electrophoresis within a gel slab chamber having separablevertical hydrophilic side walls and separable vertical end walls whichpromote a bond with a polymerized gel solution. A removable seal isplaced across the bottom end of the gel slab chamber supported byremovable means adapted to admit filling device for filling the gel slabchamber from the bottom. The vertical end walls enhancing polymerizationserve to firmly hold the gel slab within the chamber afterpolymerization when the bottom seal is removed. Ajustable means secureall of the vertical walls of the gel slab chamber together in a unitaryassembly. The gel slab chamber is placed in an outer container so thatthe lower end of the gel slab chamber is in communication with a buffersolution placed in the outer container. An upper buffer chamber is alsoplaced in the outer container in communication with the top of thevertical gel slab. The upper buffer chamber is filled with a buffersolution, and a separate electrode is placed in electrical contact withthe buffer solution in each of the upper and lower buffer chambers. Asample is underlaid beneath the upper buffer solution on top of thevertical gel slab and an electrical potential is applied between theelectrodes for causing the separation of the sample within the gel slabelectrophoretically.

In general it is an object of the present invention to provide avertical gel slab electrophoresis apparatus in which the gel slab iscast in a truly vertical position for eliminating discontinuities in thegel slab due to trapped bubbles.

It is another object of the present invention to provide a vertical gelslab electrophoresis apparatus which prevents buffer solution leakageand which holds a gel slab firmly within the chamber afterpolymerization.

It is another object of the present invention to provide a vertical gelslab electrophoresis device in which the gel slab chamber may be filledfrom the bottom for preserving the gradient in a separating gelsolution.

It is another object of the present invention to provide a vertical gelslab electrophoresis apparatus in which a plurality of samples may beseparated simultaneously under identical conditions.

It is another object of the present invention to provide a vertical gelslab electrophoresis apparatus in which the operator is protected fromelectrical shock.

It is another object of the present invention to provide a vertical gelslab electrophoresis apparatus in which hydrostatic equilibrium ismaintained across the bottom surface of the gel slab to preventdistortion therein.

It is another object of the present invention to provide a vertical gelslab electrophoresis apparatus in which the gel slab chamber isseparable for easy removal of the gel slab after electrophoresis.

It is another object of the present invention to provide a vertical gelslab electrophoresis apparatus in which the gel slab chamber is filled,the gel slab is polymerized, and the sample is separated within the gelslab without disturbing the gel slab chamber.

It is another object of the present invention to provide a vertical gelslab electrophoresis apparatus which may be used as a preparativeelectrophoresis device.

Additional objects and features of the invention will appear from thefollowing description in which the preferred embodiment has been setforth in detail in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a side elevational view of the inner container and the gelslab chambers on a leveling plate.

FIG. 2 is a plan view of the inner container and the gel slab chamberson the leveling plate.

FIG. 3 is a sectional view along the line 3--3 of FIG. 2.

FIG. 4 is a sectional view along the line 4--4 of FIG. 1.

FIG. 5 is a side elevational view of the inner container within an outercontainer and a cover poised above the top of the outer container.

FIG. 6 is a partial side elevational view showing a gel slab chambersealing cam configuration.

FIG. 7 is a sectional view along the line 7--7 of FIG. 6.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The vertical gel slab formation and electrophoresis apparatus disclosedherein provides advantages heretofore unavailable in gel slabelectrophoresis devices. Referring to FIG. 1 an inner container 11 isshown mounted atop leveling plate 12 supported at three points. Twopoints are adjustable in height by screws 13 and the third point isprovided by nonadjustable foot 14. An enclosed chamber 16 has an inlet17 and an outlet 18 for circulating a temperature control fluidtherethrough.

Referring to FIG. 2 a vertical gel slab chamber 29 of one thickness anda vertical gel slab chamber 21 of a lesser thickness is shown mounted oneither side of inner container 11 by adjustable clamps 22. Both gel slabchambers 19 and 21 have separable opposing vertical side walls 23 of ahydrophilic material such as glass. Gel slab chambers 19 and 21 alsohave separable opposing vertical end walls or spacers 24 made of amaterial which promotes a bond with a polymerized gel solution. A groove26 having a general U-shape opening upwardly has inserted therein a seal27 for contacting vertical side wall 23 nearest the vertical side ofinner container 11. It may be seen in FIG. 2 that adjustable clamps 22secure side walls 23 and end walls 24 together in a unitary assembly,and also secure the unitary assembly to the vertical sides of innercontainer 11.

Electrodes 28 and 29 are mounted atop the end walls of inner container11 and have electrical conductors 31 and 32 extending therefromrespectively.

Supporting members 33 are attached to the lower portion of innercontainer 11 by means of screws 34. A sealing gasket 36 is attached tothe top of supporting members 33 for sealing the bottom of each of gelslab chambers 19 and 21. A passage 37 is formed in a vertical directioncentrally located along supporting members 33 for providing access tosealing gasket 36 for penetration thereof by hypodermic needle 38.Sealing gasket is self sealing to maintain the bottom seal in gel slabchambers 19 and 21 when hypodermic needle 38 is removed. Tubing 39 isconnected to hypodermic needle 38 by tapered connecter 41 for bottomfilling of gel slab chambers 19 and 21 with a separating gel solution.Supporting members 33 rest on leveling plate 12 by means of resilientfeet 42 attached to the bottom thereof. Supporting members 33 also haveelongate holes 43 for allowing passage of screws 34 so that gasket 36may be firmly pressed upward against the bottom of gel slab chambers 19and 21 as hereinafter described prior to tightening screws 34 to providea positive seal for the bottoms of chambers 19 and 21. Blocks 44 areattached to the upper portion of inner container 11 to serve as stopsfor the upper edge of the walls 23 and 24 forming gel slab chambers 19and 21. Separable opposing vertical end walls 24 may have apertures 46passing therethrough, in one embodiment, which are in communication withgel slab chambers 19 and 21.

A comb 47 is provided for insertion into the top of gel slab chambers 19and 21 having teeth 48 formed thereon for casting sample wells in thetops of the gel slabs which are formed therein. A leveling device suchas a bubble level indicator 49 is provided which is placed atop comb 47or vertical side walls 23, depending upon the phase of the process.

A top wall 51 located with inner container 11, together with the endwalls, side walls and the bottom wall of inner container 11, definesenclosed chamber 16. The space above top wall 51 within extensions ofthe end walls of inner container 11 and then outer vertical side wallsof gel slab chambers 19 and 21 defines an upwardly opening upper bufferchamber 52.

Referring to FIG. 3 the inner vertical side wall 23 of both gel slabchambers 19 and 21 has a notch 53 placing the upper end of gel slabchambers 19 and 21 in communication with upper buffer chamber 52.Conductor 31 is supported internally extending through upper bufferchamber 52.

FIG. 4 clearly shows the manner in which the side walls 23 and the endwalls 24 are secured together in a unitary assembly to the sides ofinner container 11. The seal 27 in groove 26 is shown for providing aseal at the inner vertical side wall 23 for upper buffer chamber 52.Sections of the upper adjustable clamps 22 and comb 47 are omitted inFIG. 4 for clarity.

FIG. 5 shows inner container 11 positioned inside of an outer container54 having a cover 56. Cover 56 includes a pair of female connectors 57for mating with electrodes 28 and 29. Cover 56 also has holes 58 locatedtherein for passing inlet 17 and outlet 18 from enclosed chamber 16.Tubes 59 and 61 are provided to mate with inlet 17 and outlet 18respectively for carrying temperature controlling fluid to and fromenclosed chamber 16. Electrical conductors 62 are shown for providing anelectrical potential between female connectors 57 in cover 56.

The manner in which the vertical slab electrophoresis apparatus is usedfor microanalytical biochemical analysis will now be described. Asstated above, the unit accomodates two gel slabs, each of which may haveas many as twenty sample wells. Each slab is in contact with the sameupper and lower buffer chambers providing for uniformity of bufferconditions and consequent equal current distribution through the gelslabs. One of the leakproof characteristics of the unit is provided byvertical side walls 23 which are hydrophilic. Glass is a hydrophilicsubstance and has been used for side walls 23 since it forms a strongbond with the gel, minimizing band widening and streaking, and alsominimizing leakage of the buffer solution between the gel slab and theside walls 23. Seal 27 in groove 26 may be a silicone rubber gasketproviding a positive seal between the glass plates 23 and the sides ofinner container 11. From the above it may be seen that electricallyconductive paths between the buffer solution in the upper buffer chamber52 and the buffer solution contained in the outer container 54 due tobuffer solution leakage are eliminated.

The electrophoresis unit is assembled in the following manner. Innercontainer 11 is placed on its side and vertical side wall 23 havingnotch 53 in communication with upper chamber 52 is placed adjacent to aside wall of inner container 11 in contact with seal 27. A gel slabthickness is determined and appropriate thickness polyvinylchloridevertical end walls 24 are placed along the edges of the notched verticalside wall 23. The other vertical side wall 23 is placed over thevertical end walls 24 to form the four vertical walls of the gel slabchamber 19 or 21. The notch 53 in the inner vertical side wall 23 isaligned with a matching notch in the upper buffer chamber 52. Verticalside and end walls 23 and 24 are then clamped securely together and toinner container 11 by means of adjustable clamps 22 as shown in thefigures.

After securing the unitary assemblies forming gel slab chambers 19 and21 to opposite sides of inner container 11, supporting members 33carrying gaskets 36 are placed on the lower portions of inner container11 and screws 34 are inserted through elongate holes 43 to engagethreaded holes in the lower portions of inner container 11. Gaskets 36are pressed securely against the bottoms of gel slab chambers 19 and 21and screws 34 are securely tightened, thereby sealing the bottoms of gelslab chambers 19 and 21. Separate means, such as a rotatable cam 63shown in FIGS. 6 and 7, in conjunction with a pair of holes 64 in thelower extensions of the side walls on inner container 11, and a matchingpattern of elongate holes 66 in supporting members 33, may be sued togain the contact pressure between gaskets 36 and the bottoms of chambers19 and 21. Cam 63 has a centered extension 67 with a diameter fittinghole 64 projecting from an eccentric cam portion 68 attached to aturning handle 69 for rotatable cam 63. Cam portion 68 will pass throughelongate hole 66. To move members 33 toward the bottom of chambers 19and 21 to press gasket 36 firmly therebetween, extension 67 is insertedthrough elongate hole 66 and fit into hole 64. Cam portion 68 issituated in elongage hole 66 and clockwise or counterclockwise turningof handle 69 will force the surface of cam portion 68 into contact withthe wall of hole 66, moving support member 33 toward the bottom ofchambers 19 and 21.

Hypodermic needle 38 is inserted through passages 37 in supportingmembers 33 to penetrate sealing gaskets 36 and project into gel slabchambers 19 and 21. A separating gel solution is introduced into gelslab chambers 19 and 21 through the bottom thereof until the separatinggel solution approaches a level below the end of the sample well formingcomb 47. Leveling device 49 is placed on the top of vertical side walls23 and the gel slab chambers 19 and 21 are adjusted to assume a truevertical position by adjusting leveling plate 12 by means of levelingscrews 13. In this fashion gel slabs formed in chambers 19 and 21 areproduced which have a substantially constant height across the entirewidth of the slab. For pumping acrylamide gradients into gel slabchambers 19 and 21, a flow rate of 0.5 to 1.0 milliliters per minute isrecommended. Hypodermic needle 38 is removed and seal 36 self seals thepoint of penetration. An even aqueous overlayer is placed atop the lowerseparating gel solution to provide a smooth separating surface and thegel solution is polymerized. After polymerization, the overlay fluid isdrained from the top of the separating gel and the top surface rinsed.All fluid is then removed from the top surface of the separating gel.The comb 47 having a desired number of teeth 48 for forming sample wellsis then intorduced into the top of the gel slab chambers 19 and 21--leveling device 49 is placed atop comb 47 and a stacking gel is placedin the top of the gel slab chambers. A level condition should stillexist, but adjustment may be made as before.

An aqueous solution is overlaid above the stacking gel and the stackinggel is polymerized. After polymerization of the stacking gel, comb 47 isremoved and the top of the stacking gel is rinsed. At this point thesealing gasket 36 held in place by supporting members 33 are removed byremoving screws 34. Inner container 11 is then placed inside outercontainer 54. Outer container 54 is filled with buffer solution and nowconstitutes a lower buffer chamber. Upper buffer chamber 52 is alsofilled with buffer solution to the same level as outer container 54, andsamples are underlaid beneath the upper buffer solution into the samplewells formed by teeth 48 in combs 47. The lid 56 is placed upon outercontainer 54 thereby encompassing electrodes 28 and 29 with femaleconnectors 57. Electrical conductor 32 extends into the lower bufferchamber. An electrical potential is applied between conductors 62 andconsequently between electrodes 28 and 29 for separating the samples inthe sample wells electrophoretically.

After electrophoresis cover 56 is removed, inner container 11 is liftedout of outer container 54, adjustable clamps 22 are loosened and gelslab chambers 19 and 21 are separated from inner container 11. Verticalside walls 23 are pried apart and the gel slab is removed from thevertical side wall 23 upon which it rests by directing a stream of waterbetween the gel slab and the glass side wall 23.

The thickness of the gel slab is determined by the size of the separableopposing vertical end walls 24. Comb 47 is selected to have a thicknesssubstantially the same as the thickness of vertical end walls 24. Inthis fashion the operator is provided with a choice of gel thickness.

The standard comb 47 has 20 sample well forming teeth 48 which are 4millimeters wide and 15 millimeters long. The space between each toothis 2.5 millimeters in one embodiment. The teeth are designed to permitthe escape of air bubbles that would otherwise deform the shape of thesample well. As stated above the comb 47 is made of a material whichinhibits polymerization to facilitate the removal of the comb afterpolymerization without tearing the surrounding gel.

During both the polymerization and electrophoresis phases of theprocess, a coolant is circulated through the enclosed chamber 16 whichdissipates heat evenly from the inner wall 23 of both gel slab chambers19 and 21. Heat is also evenly dissipated from the upper and lowerbuffer chambers by means of the coolant circulating through enclosedchamber 16.

It may be noted by reference to FIG. 5 that outer container 54 may befilled to the same level as inner buffer chamber 52. In this fashionhydrostatic equilibrium at the bottom surface of the gel slabs inchambers 19 and 21 is achieved which prevents distortion in lowconcentration gels. It should also be noted that cover 56 carries theelectrical potential in female connectors 57 which is only connected toelectrodes 28 and 29 when cover 56 is placed atop outer container 54.This feature prevents injury from electrical shock to an operatorthrough inadvertent contact with the inner or the outer buffer solutionwith the cover removed and a high voltage applied.

The vertical gel slab electrophoresis apparatus disclosed herein has anadditional utility as a preparative electrophoresis device whenapertures 46 are formed in opposing vertical end walls 24. When theapparatus is used in this mode, gel slab chambers 19 and 21 are filledto a point just below apertures 46 with a high concentration acrylamidegel solution, for example, which forms a dense gel plug uponpolymerization. Thereafter, a non-polymerizing fluid is laid atop thelower polymerized gel to a thickness of approximately 1 millimeter. Anupper separating gel solution is carefully laid atop thenon-polymerizing fluid to a point just below teeth 48 on comb 47 asdescribed above. The upper separating gel solution is polymerized andthe remainder of the process proceeds as described above.

During electrophoresis when the apparatus is being used as a preparativeelectrophoresis device, buffer solution 1s continually circulatedthrough one aperture 46 and out of the other aperture 46 by means ofconduits (not shown) in communication therewith. The buffer solution istransmitted to a collecting device for collecting predetermined portionsof the sample after it has migrated through the upper portion of theseperating gel. Apertures 46 are not present in vertical end walls 24unless the apparatus is being used as a preparative electrophoresisdevice.

A vertical gel slab electrophoresis apparatus and method has beendisclosed which prevents leakage between the hydrophilic vertical sidewalls of the gel slab chambers and the gel slab and which provides for aplurality of samples to be electrophoresed through two separate gelslabs simultaneously under identical electrical potential andtemperature conditions. An apparatus and method is disclosed providingfor bottom filling of the gel slab chambers with separating gel solutionwhereby filling turbulence will not disturb desired separating gelgradient characteristics. Vertical end walls of the gel slab chambersare provided which promote a bond with a polymerized gel solution forretaining the gel slabs within the gel slab chambers afterelectrophoresis.

Sample well forming implements are provided which inhibit polymerizationfor easy removal of the implements after the sample wells are formedthrough polymerization. Operator protection and hydrostatic equilibriumat the bottom surface of the gel slabs is provided in the apparatus. Asingle assembly is provided for filling, polymerizing andelectrophoresis all within the same unit. Accurate temperature controlfor casting of gel slabs and electrophoresis is provided.

I claim:
 1. A method of preparing a vertical gel slab for use inelectrophoresis without disturbing the gel slab until electrophoresis iscompleted comprising the steps of forming a gel slab chamber havinghydrophilic walls, orienting the gel slab chamber in a verticaldirection, sealing the bottom of the gel slab chamber filling the gelslab chamber from the bottom with a separating gel solution while thegel slab chamber is in a vertical position, polymerizing the separatinggel solution, removing the seal from the lower end of the gel slabchamber, placing the gel slab chamber in a container with the bottom ofthe gel slab in contact with a buffer solution, placing the upper end ofthe gel slab in contact with a separate buffer solution, underlaying asample beneath the separate buffer solution above the gel slab, applyingan electrical potential between the buffer solution and the separatebuffer solution, whereby the sample is separated electrophoretically inthe gel slab.
 2. A method for producing a vertical gel slab andseparating a sample therein electrophoretically as in claim 1 togetherwith the steps of providing an even aqueous overlayer on the separatinggel prior ot polymerization, draining the aqueous overlayer afterpolymerization, inserting a sample well forming comb above thepolymerized gel slab, overlaying the polymerized gel slab with astacking gel to a level above the lower level of the well forming comb,polymerizing the stacking gel, removing the sample well forming comb,and wherein the step of underlaying a sample beneath the separate buffersolution includes placing a sample in each of a plurality of the samplewells formed by the comb, whereby a plurality of samples may beseparated electrophoretically under identical conditions.
 3. A method offorming a gel slab vertically and separating a sample thereinelecrophoretically as in claim 1 together with the step of leveling thegel slab chamber prior to polymerizing the separating gel solution sothat the gel slab formed therein has a substantially constant heightalong the width of the slab.
 4. A method of forming a gel slabvertically and separating a sample therein electrophoretically as inclaim 1 wherein the step of filling the gel slab chamber from the bottomwith a separating gel solution includes the step of stopping the fill atan intermediate level in the gel slab chamber, and wherein the step ofpolymerizing the separating gel is followed by the steps of laying ashallow layer of non-polymerizing fluid atop the lower polymerizedseparating gel, laying an upper layer os separating gel solution atopthe non-polymerizing fluid, polymerizing the upper layer of separatinggel, and flushing the space occupied by the non-polymerizing fluid witha buffer solution during electrophoresis for carrying away andcollection of desired sample components after migration through theupper layer of the separating gel, whereby a preparative electrophoresisprocess is performed.